The following is a brief description of nucleoside triphosphates. This summary is not meant to be complete but is provided only for understanding of the invention that follows. This summary is not an admission that all of the work described below is prior art to the claimed invention.
The synthesis of nucleoside triphosphates and their incorporation into nucleic acids using polymerase enzymes has greatly assisted in the advancement of nucleic acid research. The polymerase enzyme utilizes nucleoside triphosphates as precursor molecules to assemble oligonucleotides. Each nucleotide is attached by a phosphodiester bond formed through nucleophilic attack by the 3' hydroxyl group of the oligonucleotide's last nucleotide onto the 5' triphosphate of the next nucleotide. Nucleotides are incorporated one at a time into the oligonucleotide in a 5' to 3' direction. This process allows RNA to be produced and amplified from virtually any DNA or RNA templates.
Most natural polymerase enzymes incorporate standard nucleoside triphosphates into nucleic acid. For example, a DNA polymerase incorporates dATP, dTTP, dCTP, and dGTP into DNA and an RNA polymerase generally incorporates ATP, CTP, UTP, and GTP into RNA. There are however, certain polymerases that are capable of incorporating non-standard nucleoside triphosphates into nucleic acids (Joyce, 1997, PNAS 94, 1619-1622, Huang et al., Biochemistry 36, 8231-8242).
Before nucleosides can be incorporated into RNA transcripts using polymerase enzymes they must first be converted into nucleoside triphosphates which can be recognized by these enzymes. Phosphorylation of unblocked nucleosides by treatment with POCl.sub.3 and trialkyl phosphates was shown to yield nucleoside 5'-phosphorodichloridates (Yoshikawa et al., 1969, Bull. Chem. Soc. (Japan) 42, 3505). Adenosine or 2'-deoxyadenosine 5'-triphosphate was synthesized by adding an additional step consisting of treatment with excess tri-n-butylammonium pyrophosphate in DMF followed by hydrolysis (Ludwig, 1981, Acta Biochim. et Biophys. Acad. Sci. Hung. 16, 131-133).
Non-standard nucleoside triphosphates are not readily incorporated into RNA transcripts by traditional RNA polymerases. Mutations have been introduced into RNA polymerase to facilitate incorporation of deoxyribonucleotides into RNA (Sousa & Padilla, 1995, EMBO J. 14,4609-4621, Bonner et al., 1992, EMBO J. 11, 3767-3775, Bonner et al., 1994, J. Biol. Chem. 42, 25120-25128, Aurup et al., 1992, Biochemistry 31, 9636-9641).
McGee et al., International PCT Publication No. WO 95/35102, describes the incorporation of 2'-NH.sub.2 -NTP's, 2'-F-NTP's, and 2'-deoxy-2'-benzyloxyamino UTP into RNA using bacteriophage T7 polymerase.
Wieczorek et al., 1994, Bioorganic & Medicinal Chemistry Letters 4, 987-994, describes the incorporation of 7-deaza-adenosine triphosphate into an RNA transcript using bacteriophage T7 RNA polymerase.
Lin et al., 1994, Nucleic Acids Research 22, 5229-5234, reports the incorporation of 2'-NH.sub.2 -CTP and 2'-NH.sub.2 -UTP into RNA using bacteriophage T7 RNA polymerase and polyethylene glycol containing buffer. The article describes the use of the polymerase synthesized RNA for in vitro selection of aptamers to human neutrophil elastase (HNE).